Research ArticleBRAIN HEMORRHAGE

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

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Science Translational Medicine  02 Mar 2016:
Vol. 8, Issue 328, pp. 328ra29
DOI: 10.1126/scitranslmed.aac6008
  • Fig. 1. HIF-PHD inhibitors stabilize HIF-1α and abrogate hemin-induced toxicity in rat primary neurons or immortalized neuroblasts independent of iron chelation.

    (A) DFO, an iron chelator/HIF-PHD inhibitor, blocks hemin-induced toxicity in rat primary cortical neurons (PN; median effective concentration (EC50), 34.1 μM], mouse hippocampal neuroblasts (HT22; EC50, 50.8 μM), and immortalized mouse striatal neuroblasts (Q7; EC50, 26.7 μM) as measured by MTT assay. (B) CPO, an HIF-PHD inhibitor distinct from DFO, abrogates hemin-induced toxicity in primary cortical neurons (EC50, 1.4 μM), hippocampal neuroblasts (EC50, 0.9 μM), and immortalized striatal neuroblasts (EC50, 1.6 μM). (C) DHB, a distinct HIF-PHD inhibitor that does not bind to iron (11), abrogates hemin-induced toxicity in primary cortical neurons (EC50, 34.9 μM), hippocampal neuroblasts (EC50, 86.7 μM), and immortalized striatal neuroblasts (EC50, 146.1 μM). (D to K) Representative images of live [calcein-AM, green] and dead (ethidium homodimer, red) primary cortical neurons treated with (D) saline, (E) DFO (100 μM), (F) CPO (3 μM), (G) DHB (100 μM), (H) hemin (50 μM), (I) hemin (50 μM) + DFO (100 μM), (J) hemin (50 μM) + CPO (3 μM), or (K) hemin (50 μM) + DHB (100 μM). Scale bar, 100 μm. (A to C) Significance was determined by two-way analysis of variance (ANOVA) followed by Bonferroni’s comparison test, from three independent experiments. (L) DFO, DHB, and CPO stabilize HIF-1α protein in mouse primary neurons. (M) CPO (3 μM) prevents hemin-induced neuronal death, but a CPO analog (3 μM, 5342; fig. S2) without PHD inhibitory activity does not. n.s., not significant. (N) Protection by CPO is not associated with significant reductions in total iron as measured by ICP-OES. (O to R) Live/Dead staining images of cells treated with (O) saline, (P) hemin (50 μM), (Q) hemin (50 μM) + CPO (1 μM), or (R) hemin (50 μM) + CPO analog (3 μM) in mouse hippocampal neuroblasts. Scale bar, 100 μm. One-way ANOVA followed by Dunnett’s comparison test, from triplicates (M and N). All graphs show the means ± SEM. Immunoblot data are representative of three experiments.

  • Fig. 2. Molecular reduction of HIF-PHD isoforms in the mouse striatum enhances functional recovery after ICH.

    (A and B) Scheme for validating AAV8-Cre activity and selective deletion of PHD1, PHD2, and PHD3. KO, knockout. (C) Effective recombination by injection of AAV8-Cre into the mouse striatum was verified using a tdTomato floxed reporter. (D) Coronal sections of the tdTomato mouse brain revealed that tdTomato reporter expression was highest mediolaterally at coordinates corresponding to subsequent hemorrhagic stroke. Scale bars, 1 mm (C); 100 μm (D). (E) Quantitative PCR confirmed reduction of PHD1, PHD2, and PHD3 expression in the striatum of AAV8-Cre–injected mice but not in the striatum of AAV8-GFP–injected mice (E). (F) Quantitative PCR confirmed that reduction of striatal PHD expression led to increases in the HIF-dependent genes encoding VEGF and EPO in the mouse striatum. (G and H) Conditional reduction of HIF-PHDs enhances functional recovery in mice after ICH, as shown by two behavioral tasks: the corner task (G) and the tape removal task (H). Significance was determined by two-tailed t test (E and F) or two-way ANOVA with Bonferroni’s post hoc test (G and H). All graphs show the means ± SEM.

  • Fig. 3. Adaptaquin is an HIF-PHD inhibitor that can penetrate the mouse CNS.

    (A) In silico modeling predicts that adaptaquin (yellow) fits into the active site of HIF-PHDs. (B) Effect of adaptaquin on hydroxylation of a synthetic HIF peptide by recombinant HIF-PHD2, as assayed by mass spectrometry. (C and D) Adaptaquin dose-dependently inhibits HIF-PHD activity in the mouse brain as monitored by in vivo bioluminescence imaging from dorsal (C) and ventral (D) views. (E and F) Quantitative luciferase activity measurements (from pseudocolored bioluminescence) show that adaptaquin (AQ) increased the stability of ODD-luciferase in the brain and liver. (G) Increased ODD-luciferase activity in the brain was associated with increased transcription of p21waf1/cip1, a gene induced by neuronal HIF-PHD inhibition. (H) Analysis of lysates from several mouse brain regions showed an increase in ODD-luciferase reporter activity at 0, 6, 12, and 24 hours after intraperitoneal injection of adaptaquin (30 mg/kg). (I) Similar results were seen in the kidney and liver. Significance was determined by one-way ANOVA followed by Dunnett’s comparison test for vehicle compared to adaptaquin (10 or 30 mg/kg) treatment (E and F) or by two-way ANOVA with Bonferroni’s post hoc test (G to I). All graphs shows the means ± SEM.

  • Fig. 4. Adaptaquin delivered after injury reduces cell death and enhances functional recovery in rodent models of ICH.

    (A) Experimental design for delivery of adaptaquin after ICH in mice. (B) Adaptaquin chemical structure. o.d., once daily. (C and D) Adaptaquin improved behavioral deficits associated with ICH in mice undertaking two sensory tasks: the corner task (C) and the tape removal task (D). (E to G) Adaptaquin reduced neuronal degeneration as monitored by Fluoro-Jade staining (green) in the perihematomal regions of the mouse brain. Scale bar, 100 μm. White arrows highlight degenerating neurons in the ICH group (F); this was reduced by adaptaquin treatment (G). (H and I) Stereological counting of Fluoro-Jade–positive neurons confirmed that adaptaquin treatment decreased the number of degenerating neurons in the hematoma (H) and perihematoma (I) regions. Veh, vehicle. (J) Diagram illustrating adaptaquin (30 mg/kg intraperitoneally) treatment in the rat autologous blood infusion model of ICH. (K) Adaptaquin improved single-pellet reaching in rats for up to 1 month after ICH. (L) Protocol for evaluating the total concentration of iron and zinc in the mouse brain after ICH and treatment with either vehicle or adaptaquin (30 mg/kg intraperitoneally). (M) Pseudocolored brain coronal sections from mice treated with collagenase to induce ICH. (N and O) Seven days after adaptaquin treatment, total iron (N) and zinc (O) concentrations were unchanged in the mouse CNS. Significance was determined by one-way ANOVA followed by Dunnett’s comparison test for vehicle or adaptaquin treatment after ICH (H to K) or by two-way ANOVA with Bonferroni’s post hoc test (C and D). All graphs show the means ± SEM.

  • Fig. 5. Adaptaquin prevents neuronal death by suppressing ATF4-mediated expression of prodeath genes.

    (A) Adaptaquin dose-dependently protects mouse primary cortical neurons after treatment with the glutamate analog HCA. (B) Adaptaquin (1 μM) provides complete protection for 16 hours after HCA treatment. (C) Adaptaquin-mediated protection from HCA-induced oxidative stress as shown by Live/Dead imaging, Scale bar, 100μm. (D) Heat map showing the top 1000 differentially expressed genes. Shades of red represent up-regulation and shades of green represent down-regulation of gene expression. A subset of genes dysregulated by HCA treatment are corrected after treatment with adaptaquin. (E) Proportion of oxidative stress–related genes (left) whose expression was altered to more normal levels after treatment with 0.1 μM (middle) and 1 μM (right) adaptaquin. Overrepresented GO gene categories whose expression was altered by adaptaquin. (F) Overrepresented categories included genes involved in amino acid transport and regulation of programmed cell death. (G) The table shows the microarray data for ATF4-regulated genes induced by HCA and down-regulated by protective, but not by nonprotective, concentrations of adaptaquin. Values were transformed to log2 scale. (H to J) Quantitative PCR confirmed that the ATF4 target genes Trib3 (H), MTHFD2 (I), and STC2 (J) were induced by oxidative stress, and this induction was reduced by adaptaquin (1 μM). (K and L) Up-regulation of ATF4 immunostaining 7 days after ICH. ATF4 expression is shown in red, neurons are indicated by NeuN expression (green), and nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Significance was determined by two-way ANOVA followed by Bonferroni’s comparison test from three independent experiments (A) or by one-way ANOVA followed by Dunnett’s comparison test from triplicates (B and H to J). All graphs show the means ± SEM.

  • Fig. 6. Adaptaquin inhibits ATF4 DNA binding, ATF4 transcriptional activity, and ATF4 prodeath properties in neurons.

    (A) HCA-induced oxidative stress up-regulated the promoter of the ATF4 target gene Trib3; this was blocked by adaptaquin (1 μM). Treatment with nonprotective concentrations of adaptaquin (0.1 μM) or an inactive oxyquinoline analog (compound 10, 1 μM) did not impede oxidative stress–induced activation of the Trib3 promoter–luciferase reporter construct. WT, wild type. (B) Mutation of the ATF4-binding site occludes oxidative stress inducibility and sensitivity to adaptaquin (1 μM). MUT, mutant. (C) ChIP studies showed that HCA-mediated oxidative stress increased ATF4 occupancy on the Trib3 promoter, which was blocked by protective concentrations of adaptaquin (1 μM). IgG, immunoglobulin G. (D to L) MTT assay (D) or Live/Dead staining (E to L) demonstrated that overexpression of ATF4 was sufficient to induce neuronal death in the absence of HCA, whereas protective concentrations of adaptaquin (1 μM) mitigated death by ATF4 overexpression or HCA and ATF4 overexpression. (M) Proline residues mutated to alanine residues in the mouse ATF4-5P/A mutant construct are highlighted in red. (N) ATF4-5P/A mutant does not induce cell death. (O) Adaptaquin inhibited hydroxylation of ATF4. (P) In mouse neurons, treatment with adaptaquin dose-dependently stabilized HIF-1α. (Q) Adaptaquin stabilization of HIF-1α (red line) occurs at concentrations higher than those required for full protection from oxidative death (red line). Significance was determined by two-way ANOVA followed by Bonferroni’s comparison test from three independent experiments (A, B, D, and N) or by one-way ANOVA followed by Dunnett’s comparison test from triplicates (C). All graphs show the means ± SEM.

  • Fig. 7. Reduction of HIF-PHD isoforms in the mouse striatum reduces ICH-induced ATF4-dependent gene expression.

    (A to G) Expression of WT ATF4 (red), but not of ATF4-5P/A mutant (blue), through adenoviral infection of primary cortical neurons leads to time-dependent induction of Trib3 (C), CHOP (D), ATF3 (E), MTHFD2 (F), and STC2 (G). ChIP studies (B) correlate increases in Trib3 gene expression induced by WT ATF4 with occupancy at the Trib3 promoter. (H) Diagram of experimental plan to determine whether reduction of HIF-PHD isoforms can abrogate ATF4-dependent gene expression in vivo. (I to M) Effects of molecular reduction of HIF-PHD isoforms on ICH-induced expression of Trib3 (I), CHOP (J), ATF3 (K), MTHFD2 (L), and STC2 (M). Significance was determined by two-way ANOVA followed by Bonferroni’s comparison test from three independent experiments (B to G) or by one-way ANOVA followed by Dunnett’s comparison test (I to M). All graphs show the means ± SEM.

  • Fig. 8. Adaptaquin inhibition of ATF4-dependent gene expression defines a therapeutic window after ICH.

    (A) Schematic of experimental plan to examine the effect of adaptaquin on ICH-induced Trib3 expression. (B) Immunoblots of ATF4 from nuclear extracts of the mouse striatum in distinct treatment groups. (C) Quantitative PCR of Trib3 message. (D) Immunoblots of Trib3 protein. (E to G) We examined the effect of adaptaquin (30 mg/kg) delivered 6 hours after collagenase injection and then daily for 7 days on ICH-induced spatial neglect in the corner task (F) or sensory neglect in the tape removal task (G). Significance was determined by one-way ANOVA followed by Dunnett’s comparison test (C) or by two-way ANOVA with Bonferroni’s post hoc test (F and G). All graphs show the means ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/328/328ra29/DC1

    Materials and Methods

    Fig. S1. Hemin, the reactive ferric protoporphyrin IX group of hemoglobin, induces concentration-dependent death in multiple cell types.

    Fig. S2. CPO or a CPO analog (5342) differentially inhibit HIF-PHD activity, although they have a similar affinity for iron.

    Fig. S3. Conditional reduction of HIF-PHD1, HIF-PHD2, and HIF-PHD3 in the striatum does not affect ICH-induced hematoma size or brain edema.

    Fig. S4. Canonical HIF-PHD inhibitors DFO, CPO, and DHB do not stabilize ODD-luciferase in brains of mice.

    Fig. S5. Adaptaquin binds to and inhibits recombinant PHD2.

    Fig. S6. Adaptaquin does not inhibit collagenase activity in vivo.

    Fig. S7. Adaptaquin significantly reduced ICH-induced edema.

    Fig. S8. Adaptaquin’s beneficial effects on ICH outcomes are not associated with changes in core body temperature.

    Fig. S9. Reduction of HIF-1 α or HIF-2 α by RNA interference does not potentiate hemin-induced toxicity or affect the ability of chemically diverse HIF-PHD inhibitors to prevent hemin toxicity.

    Fig. S10. Hemin-induced neuronal death is abrogated by the HIF-PHD inhibitor adaptaquin.

    Fig. S11. Adaptaquin inhibits glutamate-induced mitochondrial dysfunction.

    Fig. S12. Adaptaquin is distinct from the classical antioxidant N-acetylcysteine in abrogating ATF4-mediated death.

    Fig. S13. Adaptaquin does not affect global histone acetylation or methylation or inhibit 12-lipoxygenase activities.

    Fig. S14. Adaptaquin does not affect 3-nitrotyrosine, a biomarker of oxidative stress.

    Fig. S15. Adaptaquin does not affect TET enzyme activity, a subfamily of the iron-, 2-oxoglutarate–, and oxygen-dependent dioxygenases that demethylate DNA, at concentrations where it completely abrogates neuronal death.

    Fig. S16. Adaptaquin protection is not associated with induction of HIF-dependent genes VEGF and EPO in the striatum of mice.

    Table S1. Inability of adaptaquin to influence likely off-target enzymes at concentrations where it fully protects neurons.

    References (51, 52)

  • Supplementary Material for:

    Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

    Saravanan S. Karuppagounder, Ishraq Alim, Soah J. Khim, Megan W. Bourassa, Sama F. Sleiman, Roseleen John, Cyrille C. Thinnes, Tzu-Lan Yeh, Marina Demetriades, Sandra Neitemeier, Dana Cruz, Irina Gazaryan, David W. Killilea, Lewis Morgenstern, Guohua Xi, Richard F. Keep, Timothy Schallert, Ryan V. Tappero, Jian Zhong, Sunghee Cho, Frederick R. Maxfield, Theodore R. Holman, Carsten Culmsee, Guo-Hua Fong, Yijing Su, Guo-li Ming, Hongjun Song, John W. Cave, Christopher J. Schofield, Frederick Colbourne, Giovanni Coppola, Rajiv R. Ratan*

    *Corresponding author. E-mail: rrr2001{at}med.cornell.edu

    Published 2 March 2016, Sci. Transl. Med. 8, 328ra29 (2016)
    DOI: 10.1126/scitranslmed.aac6008

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Hemin, the reactive ferric protoporphyrin IX group of hemoglobin, induces concentration-dependent death in multiple cell types.
    • Fig. S2. CPO or a CPO analog (5342) differentially inhibit HIF-PHD activity, although they have a similar affinity for iron.
    • Fig. S3. Conditional reduction of HIF-PHD1, HIF-PHD2, and HIF-PHD3 in the striatum does not affect ICH-induced hematoma size or brain edema.
    • Fig. S4. Canonical HIF-PHD inhibitors DFO, CPO, and DHB do not stabilize ODD-luciferase in brains of mice.
    • Fig. S5. Adaptaquin binds to and inhibits recombinant PHD2.
    • Fig. S6. Adaptaquin does not inhibit collagenase activity in vivo.
    • Fig. S7. Adaptaquin significantly reduced ICH-induced edema.
    • Fig. S8. Adaptaquin’s beneficial effects on ICH outcomes are not associated with changes in core body temperature.
    • Fig. S9. Reduction of HIF-1 α or HIF-2 α by RNA interference does not potentiate hemin-induced toxicity or affect the ability of chemically diverse HIF-PHD inhibitors to prevent hemin toxicity.
    • Fig. S10. Hemin-induced neuronal death is abrogated by the HIF-PHD inhibitor adaptaquin.
    • Fig. S11. Adaptaquin inhibits glutamate-induced mitochondrial dysfunction.
    • Fig. S12. Adaptaquin is distinct from the classical antioxidant N-acetylcysteine in abrogating ATF4-mediated death.
    • Fig. S13. Adaptaquin does not affect global histone acetylation or methylation or inhibit 12-lipoxygenase activities.
    • Fig. S14. Adaptaquin does not affect 3-nitrotyrosine, a biomarker of oxidative stress.
    • Fig. S15. Adaptaquin does not affect TET enzyme activity, a subfamily of the iron-, 2-oxoglutarate–, and oxygen-dependent dioxygenases that demethylate DNA, at concentrations where it completely abrogates neuronal death.
    • Fig. S16. Adaptaquin protection is not associated with induction of HIF-dependent genes VEGF and EPO in the striatum of mice.
    • Table S1. Inability of adaptaquin to influence likely off-target enzymes at concentrations where it fully protects neurons.
    • References (51, 52)

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